Enhanced azadirachtin production in neem (Azadirachta indica) callus through NaCl elicitation: Insights into differential protein regulation via shotgun proteomics.

With their remarkable bioactivity and evolving commercial importance, plant secondary metabolites (PSMs) have gained significant research interest in recent years. Plant tissue culture serves as a credible tool to examine how abiotic stresses modulate the production of PSMs, enabling clear insights into plant stress responses and the prospects for controlled synthesis of bioactive compounds. Azadirachta indica, or neem has been recognized as a repository of secondary metabolites for centuries, particularly for the compound named azadirachtin, due to its bio-pesticidal and high antioxidant properties. Introducing salt stress as an elicitor makes it possible to enhance the synthesis of secondary metabolites, specifically azadirachtin. Thus, in this research, in vitro callus cultures of neem were micro-propagated and induced with salinity stress to explore their effects on the production of azadirachtin and identify potential proteins associated with salinity stress through comparative shotgun proteomics (LCMS/MS). To induce salinity stress, 2-month-old calli were subjected to various concentrations of NaCl (0.05-1.5%) for 4 weeks. The results showed that the callus cultures were able to adapt and survive in the salinity treatments, but displayed a reduction in fresh weight as the NaCl concentration increased. Notably, azadirachtin production was significantly enhanced in the salinity treatment compared to control, where 1.5% NaCl-treated calli produced the highest azadirachtin amount (10.847 ± 0.037 mg/g DW). The proteomics analysis showed that key proteins related to primary metabolism, such as defence, energy, cell structure, redox, transcriptional and photosynthesis, were predominantly differentially regulated (36 upregulated and 93 downregulated). While a few proteins were identified as being regulated in secondary metabolism, they were not directly involved in the synthesis of azadirachtin. In conjunction with azadirachtin elicitation, salinity stress treatment could therefore be successfully applied in commercial settings for the controlled synthesis of azadirachtin and other plant-based compounds. Further complementary omics approaches can be employed to enhance molecular-level modifications, to facilitate large-scale production of bioactive compounds in the future.

Faster and lower dose imaging: evaluating adaptive, constant gantry velocity and angular separation in fast low-dose 4D cone beam CT imaging.

BACKGROUND: The adoption of four-dimensional cone beam computed tomography (4DCBCT) for image-guided lung cancer radiotherapy is increasing, especially for hypofractionated treatments. However, the drawbacks of 4DCBCT include long scan times (∼240 s), inconsistent image quality, higher imaging dose than necessary, and streaking artifacts. With the emergence of linear accelerators that can acquire 4DCBCT scans in a short period of time (9.2 s) there is a need to examine the impact that these very fast gantry rotations have on 4DCBCT image quality. PURPOSE: This study investigates the impact of gantry velocity and angular separation between x-ray projections on image quality and its implication for fast low-dose 4DCBCT with emerging systems, such as the Varian Halcyon that provide fast gantry rotation and imaging. Large and uneven angular separation between x-ray projections is known to reduce 4DCBCT image quality through increased streaking artifacts. However, it is not known when angular separation starts degrading image quality. The study assesses the impact of constant and adaptive gantry velocity and determines the level when angular gaps impair image quality using state-of-the-art reconstruction methods. METHODS: This study considers fast low-dose 4DCBCT acquisitions (60-80 s, 200-projection scans). To assess the impact of adaptive gantry rotations, the angular position of x-ray projections from adaptive 4DCBCT acquisitions from a 30-patient clinical trial were analyzed (referred to as patient angular gaps). To assess the impact of angular gaps, variable and static angular gaps (20°, 30°, 40°) were introduced into evenly separated 200 projections (ideal angular separation). To simulate fast gantry rotations, which are on emerging linacs, constant gantry velocity acquisitions (9.2 s, 60 s, 120 s, 240 s) were simulated by sampling x-ray projections at constant intervals using the patient breathing traces from the ADAPT clinical trial (ACTRN12618001440213). The 4D Extended Cardiac-Torso (XCAT) digital phantom was used to simulate projections to remove patient-specific image quality variables. Image reconstruction was performed using Feldkamp-Davis-Kress (FDK), McKinnon-Bates (MKB), and Motion-Compensated-MKB (MCMKB) algorithms. Image quality was assessed using Structural Similarity-Index-Measure (SSIM), Contrast-to-Noise-Ratio (CNR), Signal-to-Noise-Ratio (SNR), Tissue-Interface-Width-Diaphragm (TIW-D), and Tissue-Interface-Width-Tumor (TIW-T). RESULTS: Patient angular gaps and variable angular gap reconstructions produced similar results to ideal angular separation reconstructions, while static angular gap reconstructions produced lower image quality metrics. For MCMKB-reconstructions, average patient angular gaps produced SSIM-0.98, CNR-13.6, SNR-34.8, TIW-D-1.5 mm, and TIW-T-2.0 mm, static angular gap 40° produced SSIM-0.92, CNR-6.8, SNR-6.7, TIW-D-5.7 mm, and TIW-T-5.9 mm and ideal produced SSIM-1.00, CNR-13.6, SNR-34.8, TIW-D-1.5 mm, and TIW-T-2.0 mm. All constant gantry velocity reconstructions produced lower image quality metrics than ideal angular separation reconstructions regardless of the acquisition time. Motion compensated reconstruction (MCMKB) produced the highest contrast images with low streaking artifacts. CONCLUSION: Very fast 4DCBCT scans can be acquired provided that the entire scan range is adaptively sampled, and motion-compensated reconstruction is performed. Importantly, the angular separation between x-ray projections within each individual respiratory bin had minimal effect on the image quality of fast low-dose 4DCBCT imaging. The results will assist the development of future 4DCBCT acquisition protocols that can now be achieved in very short time frames with emerging linear accelerators.

Geographical Diversity of Proteomic Responses to Cold Stress in the Fungal Genus Pseudogymnoascus.

In understanding stress response mechanisms in fungi, cold stress has received less attention than heat stress. However, cold stress has shown its importance in various research fields. The following study examined the cold stress response of six Pseudogymnoascus spp. isolated from various biogeographical regions through a proteomic approach. In total, 2541 proteins were identified with high confidence. Gene Ontology enrichment analysis showed diversity in the cold stress response pathways for all six Pseudogymnoascus spp. isolates, with metabolic and translation-related processes being prominent in most isolates. 25.6% of the proteins with an increase in relative abundance were increased by more than 3.0-fold. There was no link between the geographical origin of the isolates and the cold stress response of Pseudogymnoascus spp. However, one Antarctic isolate, sp3, showed a distinctive cold stress response profile involving increased flavin/riboflavin biosynthesis and methane metabolism. This Antarctic isolate (sp3) was also the only one that showed decreased phospholipid metabolism in cold stress conditions. This work will improve our understanding of the mechanisms of cold stress response and adaptation in psychrotolerant soil microfungi, with specific attention to the fungal genus Pseudogymnoascus.

Structural insights into coordinating 5S RNP rotation with ITS2 pre-RNA processing during ribosome formation.

The rixosome defined in Schizosaccharomyces pombe and humans performs diverse roles in pre-ribosomal RNA processing and gene silencing. Here, we isolate and describe the conserved rixosome from Chaetomium thermophilum, which consists of two sub-modules, the sphere-like Rix1-Ipi3-Ipi1 and the butterfly-like Las1-Grc3 complex, connected by a flexible linker. The Rix1 complex of the rixosome utilizes Sda1 as landing platform on nucleoplasmic pre-60S particles to wedge between the 5S rRNA tip and L1-stalk, thereby facilitating the 180° rotation of the immature 5S RNP towards its mature conformation. Upon rixosome positioning, the other sub-module with Las1 endonuclease and Grc3 polynucleotide-kinase can reach a strategic position at the pre-60S foot to cleave and 5' phosphorylate the nearby ITS2 pre-rRNA. Finally, inward movement of the L1 stalk permits the flexible Nop53 N-terminus with its AIM motif to become positioned at the base of the L1-stalk to facilitate Mtr4 helicase-exosome participation for completing ITS2 removal. Thus, the rixosome structure elucidates the coordination of two central ribosome biogenesis events, but its role in gene silencing may adapt similar strategies.

Quantitative proteomics analysis of permethrin and temephos-resistant Ae. aegypti revealed diverse differentially expressed proteins associated with insecticide resistance from Penang Island, Malaysia.

Synthetic insecticides are the primary vector control method used globally. However, the widespread use of insecticides is a major cause of insecticide-resistance in mosquitoes. Hence, this study aimed at elucidating permethrin and temephos-resistant protein expression profiles in Ae. aegypti using quantitative proteomics. In this study, we evaluated the susceptibility of Ae. aegypti from Penang Island dengue hotspot and non-hotspot against 0.75% permethrin and 31.25 mg/l temephos using WHO bioassay method. Protein extracts from the mosquitoes were then analysed using LC-ESI-MS/MS for protein identification and quantification via label-free quantitative proteomics (LFQ). Next, Perseus 1.6.14.0 statistical software was used to perform differential protein expression analysis using ANOVA and Student's t-test. The t-test selected proteins with≥2.0-fold change (FC) and ≥2 unique peptides for gene expression validation via qPCR. Finally, STRING software was used for functional ontology enrichment and protein-protein interactions (PPI). The WHO bioassay showed resistance with 28% and 53% mortalities in adult mosquitoes exposed to permethrin from the hotspot and non-hotspot areas. Meanwhile, the susceptibility of Ae. aegypti larvae revealed high resistance to temephos in hotspot and non-hotspot regions with 80% and 91% mortalities. The LFQ analyses revealed 501 and 557 (q-value <0.05) differentially expressed proteins in adults and larvae Ae. aegypti. The t-test showed 114 upregulated and 74 downregulated proteins in adult resistant versus laboratory strains exposed to permethrin. Meanwhile, 13 upregulated and 105 downregulated proteins were observed in larvae resistant versus laboratory strains exposed to temephos. The t-test revealed the upregulation of sodium/potassium-dependent ATPase ß2 in adult permethrin resistant strain, H15 domain-containing protein, 60S ribosomal protein, and PB protein in larvae temephos resistant strain. The downregulation of troponin I, enolase phosphatase E1, glucosidase 2ß was observed in adult permethrin resistant strain and tubulin ß chain in larvae temephos resistant strain. Furthermore, the gene expression by qPCR revealed similar gene expression patterns in the above eight differentially expressed proteins. The PPI of differentially expressed proteins showed a p-value at <1.0 x 10-16 in permethrin and temephos resistant Ae. aegypti. Significantly enriched pathways in differentially expressed proteins revealed metabolic pathways, oxidative phosphorylation, carbon metabolism, biosynthesis of amino acids, glycolysis, and citrate cycle. In conclusion, this study has shown differentially expressed proteins and highlighted upregulated and downregulated proteins associated with insecticide resistance in Ae. aegypti. The validated differentially expressed proteins merit further investigation as a potential protein marker to monitor and predict insecticide resistance in field Ae. aegypti. The LC-MS/MS data were submitted into the MASSIVE database with identifier no: MSV000089259.

Mechanism of 5S RNP recruitment and helicase-surveilled rRNA maturation during pre-60S biogenesis.

Ribosome biogenesis proceeds along a multifaceted pathway from the nucleolus to the cytoplasm that is extensively coupled to several quality control mechanisms. However, the mode by which 5S ribosomal RNA is incorporated into the developing pre-60S ribosome, which in humans links ribosome biogenesis to cell proliferation by surveillance by factors such as p53-MDM2, is poorly understood. Here, we report nine nucleolar pre-60S cryo-EM structures from Chaetomium thermophilum, one of which clarifies the mechanism of 5S RNP incorporation into the early pre-60S. Successive assembly states then represent how helicases Dbp10 and Spb4, and the Pumilio domain factor Puf6 act in series to surveil the gradual folding of the nearby 25S rRNA domain IV. Finally, the methyltransferase Spb1 methylates a universally conserved guanine nucleotide in the A-loop of the peptidyl transferase center, thereby licensing further maturation. Our findings provide insight into the hierarchical action of helicases in safeguarding rRNA tertiary structure folding and coupling to surveillance mechanisms that culminate in local RNA modification.

Fourth-Generation Minimally Invasive Hallux Valgus Surgery With Metaphyseal Extra-Articular Transverse and Akin Osteotomy (META): 12 Month Clinical and Radiologic Results.

BACKGROUND: Fourth-generation minimally invasive surgery (MIS) includes the multiplanar rotational deformity correction achieved through manipulation of an extra-articular distal first metatarsal osteotomy that is held with rigid fixation using 2 fully threaded screws, of which one must be bicortical to provide rotational and biomechanical stability. The aim of this study is to report the clinical and radiologic outcomes of an evolved fourth-generation MIS hallux valgus technique. METHODS: A prospective single-surgeon series of consecutive patients undergoing fourth-generation MIS was performed using a distal transverse osteotomy with a minimum 12-month follow-up. The primary outcome was the Manchester-Oxford Foot Questionnaire (MOXFQ), a validated clinical patient-reported outcome measure (PROM). Secondary outcomes included radiographic deformity correction, clinical assessment, and EuroQol-5D-5L PROMs. RESULTS: Between September 2019 and June 2021, 50 feet underwent fourth-generation MIS. The mean age was 55.8±15.3 years with a mean follow-up of 1.4 years. Preoperative and minimum 12-month primary outcome data were available for 100% of feet. There was a significant improvement in all MOXFQ domain scores, with the index domain improving from 53.4 to 13.1 (P < .001). There was a significant improvement (P < .001) in hallux valgus angle (32.7 to 7.9 degrees), intermetatarsal angle (14.0 to 4.2 degrees) and distal metatarsal articular angle (18.5 to 5.6 degrees). There was a significant improvement in general health-related quality of life EQ-5D-5L index and EQ-VAS scores (P < .05). CONCLUSION: The fourth-generation MIS technique is a safe and effective approach to hallux valgus deformity correction with significant improvement in clinical and radiographic outcomes. LEVEL OF EVIDENCE: Level IV, prospective case series.

Cross-reactivity analysis of milk proteins from different goat breeds with cow's milk allergens using a proteomic approach.

INTRODUCTION: Goat's milk thought to be a good substitute for cow's milk protein allergic (CMPA) individuals. However, there is growing evidence that their proteins have cross-reactivities with cow's milk allergens. This study aimed to profile and compare milk proteins from different goat breeds that have cross-reactivity to cow's milk allergens. METHODOLOGY: Proteomics was used to compare protein extracts of skim milk from Saanen, Jamnapari, and Toggenburg. Cow's milk was used as a control. IgE-immunoblotting and mass spectrometry were used to compare and identify proteins that cross-reacted with serum IgE from CMPA patients (n = 10). RESULTS: The analysis of IgE-reactive proteins revealed that the protein spots identified with high confidence were proteins homologous to common cow's milk allergens such as α-S1-casein (αS1-CN), ß-casein (ß-CN), κ-casein (κ-CN), and beta-lactoglobulin (ß-LG). Jamnapari's milk proteins were found to cross-react with four major milk allergens: α-S1-CN, ß-CN, κ-CN, and ß-LG. Saanen goat's milk proteins, on the other hand, cross-reacted with two major milk allergens, α-S1-CN and ß-LG, whereas Toggenburg goat's milk proteins only react with one of the major milk allergens, κ-CN. CONCLUSION: These findings may help in the development of hypoallergenic goat milk through cross-breeding strategies of goat breeds with lower allergenic milk protein contents.

The nucleoplasmic phase of pre-40S formation prior to nuclear export.

Biogenesis of the small ribosomal subunit in eukaryotes starts in the nucleolus with the formation of a 90S precursor and ends in the cytoplasm. Here, we elucidate the enigmatic structural transitions of assembly intermediates from human and yeast cells during the nucleoplasmic maturation phase. After dissociation of all 90S factors, the 40S body adopts a close-to-mature conformation, whereas the 3' major domain, later forming the 40S head, remains entirely immature. A first coordination is facilitated by the assembly factors TSR1 and BUD23-TRMT112, followed by re-positioning of RRP12 that is already recruited early to the 90S for further head rearrangements. Eventually, the uS2 cluster, CK1 (Hrr25 in yeast) and the export factor SLX9 associate with the pre-40S to provide export competence. These exemplary findings reveal the evolutionary conserved mechanism of how yeast and humans assemble the 40S ribosomal subunit, but reveal also a few minor differences.

Cannabinoids and Opioids Differentially Target Extrinsic and Intrinsic GABAergic Inputs onto the Periaqueductal Grey Descending Pathway.

The midbrain periaqueductal gray (PAG) plays a central role in pain modulation via descending pathways. Opioids and cannabinoids are thought to activate these descending pathways by relieving intrinsic GABAergic inhibition of PAG neurons which project to the rostroventromedial medulla (RVM), a process known as disinhibition. However, the PAG also receives descending extrinsic GABAergic inputs from the central nucleus of the amygdala (CeA) which are thought to inhibit PAG GABAergic interneurons. It remains unclear how opioids and cannabinoids act at these different synapses to control descending analgesic pathways. We used optogenetics, tract tracing and electrophysiology to identify the circuitry underlying opioid and cannabinoid actions within the PAG of male and female rats. It was observed that both RVM-projection and nonprojection PAG neurons received intrinsic-PAG and extrinsic-CeA synaptic inputs, which were predominantly GABAergic. Opioids acted via presynaptic µ-receptors to suppress both intrinsic and extrinsic GABAergic inputs onto all PAG neurons, although this inhibition was greater in RVM-projection neurons. By contrast, cannabinoids acted via presynaptic CB1 receptors to exclusively suppress the direct descending GABAergic input from the CeA onto RVM-projection PAG neurons. These findings indicate the CeA controls PAG output neurons which project to the RVM via parallel direct and indirect GABAergic pathways. While µ-opioids indiscriminately inhibit GABAergic inputs onto all PAG neurons, cannabinoids selectively inhibit a direct extrinsic GABAergic input from the amygdala onto PAG projection neurons. These differential actions of opioids and cannabinoids provide a flexible system to gate the descending control of analgesia from the PAG.SIGNIFICANCE STATEMENT The disinhibition hypothesis of analgesia states that opioids activate the midbrain periaqueductal gray (PAG) descending pathway by relieving the tonic inhibition of projection neurons from GABAergic interneurons. However, the PAG also receives extrinsic GABAergic inputs and is the locus of action of cannabinoid analgesics. Here, we show the relative sensitivity of GABAergic synapses to opioids and cannabinoids within the PAG depends on both the origin of presynaptic inputs and their postsynaptic targets. While opioids indiscriminately inhibit all GABAergic inputs onto all PAG neurons, cannabinoids selectively inhibit a direct extrinsic GABAergic input from the amygdala onto PAG descending projection neurons. These differential actions of opioids and cannabinoids provide a flexible system to gate PAG descending outputs.

Cms1 coordinates stepwise local 90S pre-ribosome assembly with timely snR83 release.

Ribosome synthesis begins in the nucleolus with 90S pre-ribosome construction, but little is known about how the many different snoRNAs that modify the pre-rRNA are timely guided to their target sites. Here, we report a role for Cms1 in such a process. Initially, we discovered CMS1 as a null suppressor of a nop14 mutant impaired in Rrp12-Enp1 factor recruitment to the 90S. Further investigations detected Cms1 at the 18S rRNA 3' major domain of an early 90S that carried H/ACA snR83, which is known to guide pseudouridylation at two target sites within the same subdomain. Cms1 co-precipitates with many 90S factors, but Rrp12-Enp1 encircling the 3' major domain in the mature 90S is decreased. We suggest that Cms1 associates with the 3' major domain during early 90S biogenesis to restrict premature Rrp12-Enp1 binding but allows snR83 to timely perform its modification role before the next 90S assembly steps coupled with Cms1 release take place.

Computationally Designed Anti-LuxP DNA Aptamer Suppressed Flagellar Assembly- and Quorum Sensing-Related Gene Expression in Vibrio parahaemolyticus.

(1) Background: Quorum sensing (QS) is the chemical communication between bacteria that sense chemical signals in the bacterial population to control phenotypic changes through the regulation of gene expression. The inhibition of QS has various potential applications, particularly in the prevention of bacterial infection. QS can be inhibited by targeting the LuxP, a periplasmic receptor protein that is involved in the sensing of the QS signaling molecule known as the autoinducer 2 (AI-2). The sensing of AI-2 by LuxP transduces the chemical information through the inner membrane sensor kinase LuxQ protein and activates the QS cascade. (2) Methods: An in silico approach was applied to design DNA aptamers against LuxP in this study. A method combining molecular docking and molecular dynamics simulations was used to select the oligonucleotides that bind to LuxP, which were then further characterized using isothermal titration calorimetry. Subsequently, the bioactivity of the selected aptamer was examined through comparative transcriptome analysis. (3) Results: Two aptamer candidates were identified from the ITC, which have the lowest dissociation constants (Kd) of 0.2 and 0.5 micromolar. The aptamer with the lowest Kd demonstrated QS suppression and down-regulated the flagellar-assembly-related gene expression. (4) Conclusions: This study developed an in silico approach to design an aptamer that possesses anti-QS properties.

In vitro structural maturation of an early stage pre-40S particle coupled with U3 snoRNA release and central pseudoknot formation.

The transition of the 90S to the pre-40S pre-ribosome is a decisive step in eukaryotic small subunit biogenesis leading to a first pre-40S intermediate (state Dis-C or primordial pre-40S), where the U3 snoRNA keeps the nascent 18S rRNA locally immature. We in vitro reconstitute the ATP-dependent U3 release from this particle, catalyzed by the helicase Dhr1, and follow this process by cryo-EM revealing two successive pre-40S intermediates, Dis-D and Dis-E. The latter has lost not only U3 but all residual 90S factors including the GTPase Bms1. In vitro remodeling likewise induced the formation of the central pseudoknot, a universally conserved tertiary RNA structure that comprises the core of the small subunit decoding center. Thus, we could structurally reveal a key tertiary RNA folding step that is essential to form the active 40S subunit.

Reducing 4DCBCT imaging dose and time: exploring the limits of adaptive acquisition and motion compensated reconstruction.

This study investigates the dose and time limits of adaptive 4DCBCT acquisitions (adaptive-acquisition) compared with current conventional 4DCBCT acquisition (conventional-acquisition). We investigate adaptive-acquisitions as low as 60 projections (∼25 s scan, 6 projections per respiratory phase) in conjunction with emerging image reconstruction methods. 4DCBCT images from 20 patients recruited into the adaptive CT acquisition for personalized thoracic imaging clinical study (NCT04070586) were resampled to simulate faster and lower imaging dose acquisitions. All acquisitions were reconstructed using Feldkamp-Davis-Kress (FDK), McKinnon-Bates (MKB), motion compensated FDK (MCFDK), motion compensated MKB (MCMKB) and simultaneous motion estimation and image reconstruction (SMEIR) algorithms. All reconstructions were compared against conventional-acquisition 4DFDK-reconstruction using Structural SIMilarity Index (SSIM), signal-to-noise ratio (SNR), contrast-to-noise-ratio (CNR), tissue interface sharpness diaphragm (TIS-D), tissue interface sharpness tumor (TIS-T) and center of mass trajectory (COMT) for difference in diaphragm and tumor motion. All reconstruction methods using 110-projection adaptive-acquisition (11 projections per respiratory phase) had a SSIM of greater than 0.92 relative to conventional-acquisition 4DFDK-reconstruction. Relative to conventional-acquisition 4DFDK-reconstruction, 110-projection adaptive-acquisition MCFDK-reconstructions images had 60% higher SNR, 10% higher CNR, 30% higher TIS-T and 45% higher TIS-D on average. The 110-projection adaptive-acquisition SMEIR-reconstruction images had 123% higher SNR, 90% higher CNR, 96% higher TIS-T and 60% higher TIS-D on average. The difference in diaphragm and tumor motion compared to conventional-acquisition 4DFDK-reconstruction was within submillimeter accuracy for all acquisition reconstruction methods. Adaptive-acquisitions resulted in faster scans with lower imaging dose and equivalent or improved image quality compared to conventional-acquisition. Adaptive-acquisition with motion compensated-reconstruction enabled scans with as low as 110 projections to deliver acceptable image quality. This translates into 92% lower imaging dose and 80% less scan time than conventional-acquisition.

Protein homeostasis, regulation of energy production and activation of DNA damage-repair pathways are involved in the heat stress response of Pseudogymnoascus spp.

Proteome changes can be used as an instrument to measure the effects of climate change, predict the possible future state of an ecosystem and the direction in which is headed. In this study, proteomic and gene ontology functional enrichment analysis of six Pseudogymnoascus spp. isolated from various global biogeographical regions were carried out to determine their response to heat stress. In total, 2122 proteins were identified with high confidence. Comparative quantitative analysis showed that changes in proteome profiles varied greatly between isolates from different biogeographical regions. Although the identities of the proteins that changed varied between the different regions, the functions they governed were similar. Gene ontology analysis showed enrichment of proteins involved in multiple protective mechanisms, including the modulation of protein homeostasis, regulation of energy production and activation of DNA damage and repair pathways. Our proteomic analysis did not show any clear relationship between protein changes and the strains' biogeographical origins.

Endogenous S-nitrosocysteine proteomic inventories identify a core of proteins in heart metabolic pathways.

Protein cysteine residues are essential for protein folding, participate in enzymatic catalysis, and coordinate the binding of metal ions to proteins. Enzymatically catalyzed and redox-dependent post-translational modifications of cysteine residues are also critical for signal transduction and regulation of protein function and localization. S-nitrosylation, the addition of a nitric oxide equivalent to a cysteine residue, is a redox-dependent modification. In this study, we curated and analyzed four different studies that employed various chemoselective platforms coupled to mass spectrometry to precisely identify S-nitrosocysteine residues in mouse heart proteins. Collectively 1974 S-nitrosocysteine residues in 761 proteins were identified and 33.4% were identified in two or more studies. A core of 75 S-nitrosocysteine residues in 44 proteins were identified in all four studies. Bioinformatic analysis of each study indicated a significant enrichment of mitochondrial proteins participating in metabolism. Regulatory proteins in glycolysis, TCA cycle, oxidative phosphorylation and ATP production, long chain fatty acid ß-oxidation, and ketone and amino acid metabolism constitute the major functional pathways impacted by protein S-nitrosylation. In the cardiovascular system, nitric oxide signaling regulates vasodilation and cardiac muscle contractility. The meta-analysis of the proteomic data supports the hypothesis that nitric oxide signaling via protein S-nitrosylation is also a regulator of cardiomyocyte metabolism that coordinates fuel utilization to maximize ATP production. As such, protein cysteine S-nitrosylation represents a third functional dimension of nitric oxide signaling in the cardiovascular system to ensure optimal cardiac function.

Obesity-induced astrocyte dysfunction impairs heterosynaptic plasticity in the orbitofrontal cortex.

Overconsumption of highly palatable, energy-dense food is considered a key driver of the obesity pandemic. The orbitofrontal cortex (OFC) is critical for reward valuation of gustatory signals, yet how the OFC adapts to obesogenic diets is poorly understood. Here, we show that extended access to a cafeteria diet impairs astrocyte glutamate clearance, which leads to a heterosynaptic depression of GABA transmission onto pyramidal neurons of the OFC. This decrease in GABA tone is due to an increase in extrasynaptic glutamate, which acts via metabotropic glutamate receptors to liberate endocannabinoids. This impairs the induction of endocannabinoid-mediated long-term plasticity. The nutritional supplement, N-acetylcysteine rescues this cascade of synaptic impairments by restoring astrocytic glutamate transport. Together, our findings indicate that obesity targets astrocytes to disrupt the delicate balance between excitatory and inhibitory transmission in the OFC.

The first-in-human implementation of adaptive 4D cone beam CT for lung cancer radiotherapy: 4DCBCT in less time with less dose.

BACKGROUND AND PURPOSE: We present the first implementation of Adaptive 4D cone beam CT (4DCBCT) that adapts the image hardware (gantry rotation speed and kV projections) in response to the patient's real-time respiratory signal. Adaptive 4DCBCT was applied on lung cancer patients to reduce the scan time and imaging dose in the ADaptive CT Acquisition for Personalised Thoracic imaging (ADAPT) trial. MATERIALS AND METHODS: The ADAPT technology measures the patient's real-time respiratory signal and uses mathematical optimisation and external circuitry attached to the linear accelerator to modulate the gantry rotation speed and kV projection rate to reduce scan times and imaging dose. For each patient, ADAPT scans were acquired on two treatment fractions and reconstructed with a motion compensated reconstruction algorithm and compared to the current state-of-the-art four-minute 4DCBCT acquisition (conventional 4DCBCT). We report on the scan time, imaging dose and image quality for the first four adaptive 4DCBCT patients. RESULTS: The ADAPT imaging dose was reduced by 85% and scan times were 73 ± 12 s representing a 70% reduction compared to the 240 s conventional 4DCBCT scan. The contrast-to-noise ratio was improved from 9.2 ± 3.9 with conventional 4DCBCT to 11.7 ± 4.1 with ADAPT. DISCUSSION: The ADAPT trial represents the first time that gantry rotation speed and projection acquisition have been adapted and optimised in real-time in response to changes in the patient's breathing. ADAPT demonstrates substantially reduced scan times and imaging dose for clinical 4DCBCT imaging that could enable more efficient and optimised lung cancer radiotherapy.

Reducing 4DCBCT scan time and dose through motion compensated acquisition and reconstruction.

Conventional 4DCBCT captures 1320 projections across 4 min. Adaptive 4DCBCT has been developed to reduce imaging dose and scan time. This study investigated reconstruction algorithms that best complement adaptive 4DCBCT acquisition for reducing imaging dose and scan time whilst maintaining or improving image quality compared to conventional 4DCBCT acquisition using real patient data from the first 10 adaptive 4DCBCT patients. Adaptive 4DCBCT was implemented in the ADaptive CT Acquisition for Personalized Thoracic imaging clinical trial. Adaptive 4DCBCT modulates gantry rotation speed and kV acquisition rate in response to the patient's real-time respiratory signal, ensuring even angular spacing between projections at each respiratory phase. We examined the first 10 lung cancer radiotherapy patients that received adaptive 4DCBCT. Fast, 200-projection scans over 60-80 s, and slower, 600-projection scans over ∼240 s, were obtained after routine patient treatment and compared against conventional 4DCBCT acquisition. Adaptive 4DCBCT acquisitions were reconstructed using Feldkamp-Davis-Kress (FDK), McKinnon-Bates (MKB), Motion Compensated FDK (MCFDK) and Motion Compensated MKB (MCMKB) algorithms. Reconstructions were assessed via, Structural SIMilarity (SSIM), Signal-to-Noise-Ratio (SNR), Contrast-to-Noise-Ratio (CNR), Tissue Interface Sharpness of Diaphragm (TIS-D) and Tumor (TIS-T). The 200- and 600-projection adaptive 4DCBCT acquisition corresponded to 85% and 55% reduction in imaging dose, shorter and similar scan times of approximately 90 s and 236 s respectively, compared to conventional 4DCBCT acquisition. 200- and 600-projection adaptive 4DCBCT reconstructions achieved more than 0.900 SSIM relative to conventional 4DCBCT acquisition. Compared to conventional 4DCBCT acquisition, 200-projection adaptive 4DCBCT reconstructions achieved higher SNR, CNR, TIS-T, TIS-D with motion compensated algorithms, MCFDK (208%, 159%, 174%, 247%) and MCMKB (214%, 173%, 266%, 245%) respectively. The 200-projection adaptive 4DCBCT MCFDK- and MCMKB-reconstruction results show image quality improvements are possible even with 85% fewer projections acquired. We established acquisition-reconstruction protocols that provide substantial reductions in imaging time and dose whilst improving image quality.

Transcriptomic and proteomic profiling revealed reprogramming of carbon metabolism in acetate-grown human pathogen Candida glabrata.

BACKGROUND: Emergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection. METHODS: In this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches. RESULTS: Collectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages. CONCLUSION: Here, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host.